1. The Field of the Invention
The present invention relates to techniques for optically measuring levels of chemical compounds in biological tissues. More particularly, the invention relates to the noninvasive optical detection and measurement of levels of macular carotenoids and related chemical substances using spectrally selective fluorescence spectroscopy of lipofuscin.
2. The Relevant Technology
Macular pigment (“MP”) is a collection of biological compounds concentrated in a small region in the center of the retina that provides high-acuity vision. Comprised of the carotenoid compounds lutein and zeaxanthin, MP is thought to play a protective role in the prevention or delay of age-related macular degeneration (“AMD”), the leading cause of irreversible blindness in the elderly in the Western world. Epidemiological studies analyzing carotenoid levels via dietary surveys and serum assays have shown that there is an inverse correlation between high dietary intakes and blood levels of lutein and zeaxanthin and risk of advanced AMD. Furthermore, several studies, including resonance Raman clinical studies and a high performance liquid chromatography (“HPLC”) analysis of human cadaver eyes with and without a known history of AMD, have demonstrated a correlation between levels of lutein and zeaxanthin and AMD.
The standard methods that have been used for measuring carotenoids are through high-performance liquid chromatography (HPLC) techniques. Such techniques require that large amounts of tissue sample be removed from the patient for subsequent analysis and processing, which typically takes at least 24 hours to complete. In the course of these types of analyses, the tissue is damaged, if not completely destroyed. As a result, there is a strong interest to develop non-invasive detection techniques for MP in the living human retina. Such techniques could be used, for example, in large-scale monitoring studies of dietary and/or nutritional interventions designed to raise MP levels, and potentially help protect a large fraction of the population from developing this debilitating disease.
Currently, the most commonly used noninvasive method for measuring human MP levels is a subjective psychophysical heterochromatic flicker photometry test involving color intensity matching of a light beam aimed at the fovea and another aimed at the perifoveal area. However, this method is rather time consuming and requires an alert, cooperative subject with good visual acuity. This method can also exhibit a high intrasubject variability when macular pigment densities are low or if significant macular pathology is present. Thus, the usefulness of this method for assessing macular pigment levels in the elderly population most at risk for AMD is severely limited. Nevertheless, researchers have used flicker photometry to investigate important questions such as variation of macular pigment density with age and diet.
A number of objective techniques for the measurement of MP in the human retina have been explored recently as alternatives to the subjective psychophysical tests. The underlying optics principles of these techniques are either based on fundus reflection or fundus fluorescence (autofluorescence) spectroscopy.
One of the MP imaging approaches is based on fundus reflectance techniques. There are two variants in which this method is implemented. In one variant, the reflectance of a broad band light source from the sclera is compared for a foveal and perifoveal spot and the spectral contribution of the absorbance by MP is calculated. In a second variant, no reference at a peripheral site is needed. Only a foveal field is used, and MP levels are derived from a model fit that takes into account the absorption and scattering coefficients of all retinal layers traversed in a double-path succession by the light.
Some reflectance based imaging variants are based on scanning laser opthalmoscopes (SLOs). Argon laser lines at 488 and 514 nm are used to generate monochromatic digital reflection images from the retina at MP “on peak” and “off peak” spectral absorption positions, which are then digitally subtracted to display the MP absorption distribution. While reflectance based MP imaging is evolving as a viable clinical technique for subjects, a drawback of the technology is seen in the need for eyes in mydriasis (dilation of the pupil).
In autofluorescence spectroscopy, lipofuscin in the retinal pigment epithelium is excited with light within and outside the wavelength range of macular pigment absorption, but within the absorption range of lipofuscin. This can be realized, for example, with 488 nm and 532 nm light sources, respectively. The blue (488 nm) wavelength is absorbed both by macular pigment and lipofuscin; the green (532 nm) wavelength is absorbed only by lipofuscin. By measuring the lipofuscin fluorescence intensity levels for the foveal and peripheral retina regions, Imin and IImax, respectively, for both excitation wavelengths, an estimate of the single-pass absorption of MP can be obtained. A disadvantage of the autofluorescence technique is its low specificity. In principle, any absorber absorbing in the same wavelength range as the MP can artifactually attenuate the lipofuscin excitation, and thus lead to an erroneous mapping of the MP distribution and its concentration levels. This could be a serious drawback, particularly in the presence of retinal pathology (e.g. drusen, bleeding vessels, etc). Similarly, fluorescence from other compounds than lipofuscin could confound the results.
MP usually peaks in the center of the macula, the foveola, and drops off rapidly with increasing eccentricity. Absolute concentrations of MP are very high compared to other tissue sites, corresponding typically to 10-30 ng per macular punch biopsy (about 5 mm diameter). FIG. 1 illustrates the absorption spectrum of an excised, flat-mounted, human retina in the blue/green wavelength region, showing typical absorption characteristics of carotenoid macular pigment (solid curve at left). The retinal pigment epithelium of the retina was removed for this measurement, and the spectrum was measured through a 1 mm aperture. In spite of the very thin retinal tissue layer, the optical density reaches an average value of about 0.3 above background, which explains the origin of the strong yellow coloration of the macula. Comparing the optical absorption of the macula with lutein and zeaxanthin solutions, one finds that the absorption behavior is remarkably similar, including the appearance of vibronic substructure, and that there is little overlap with potentially confounding other chromophores in the intact retina. The solid curve at right shows the fluorescence spectrum of a solution of lutein, obtained under excitation at 488 nm.
Optical excitation of MP leads to only very weak fluorescence since the excited lutein and zeaxanthin molecules relax very rapidly (within 200-250 fsec) to a lower lying excited state from which emission of light is parity forbidden (see FIG. 1C). The unusual ordering of the energy states is a unique feature of the polyene-like, .pi.-conjugated carotenoids having a large number of conjugated C.dbd.C double bonds (10 and 11 in lutein and zeaxanthin, respectively, see FIG. 1B). In fact, the quantum efficiency for a radiative transition from the 21Ag excited state to the 11Ag ground state is estimated to be as low as 10−5 to 10−4. Therefore, relaxation of the excited molecule back to the ground state occurs mostly via non-radiative transitions. The weak emission, observable only with very sensitive detection, has a small Stokes shift, and occurs in the green wavelength range centered at about 530 nm, as shown in FIG. 1 (solid curve at right).
Due to the weak fluorescence transitions, direct detection of MP using lutein or zeaxanthin fluorescence has not been realized to date. However, the virtual absence of intrinsic MP fluorescence makes it possible to detect instead the resonance Raman transitions of MP, even in living human eyes, which would otherwise be masked beyond detection by the fluorescence background. As a result, one method for the measurement of carotenoids and related chemical substances in biological tissue is by resonance Raman spectroscopy, for example as disclosed in U.S. Pat. No. 6,205,354, the disclosure of which is incorporated by reference herein in its entirety. Generally, Raman spectroscopy is a highly specific form of vibrational spectroscopy that identifies a Raman shift, which corresponds to an energy which is the fingerprint of the vibrational or rotational energy state of certain molecules. Typically, a molecule exhibits several characteristic Raman active vibrational or rotational energy states, and the measurement of the molecule's Raman spectrum thus provides a fingerprint of the molecule, i.e., it provides a molecule-specific series of spectrally sharp vibration or rotation peaks. The intensity of the Raman scattered light corresponds directly to the concentration of the molecule(s) of interest. In the case of Raman spectroscopy as applied to MP, this method detects the light that is Raman scattered from the MP carotenoid molecules at their 1525 cm.sup.-1 carbon-carbon double bond stretch frequency under resonant excitation in the MP absorption band. The Raman method measures the response of MP directly and has a very high molecule specificity.
Raman detection methods have the benefit of extremely high specificity for lutein and zeaxanthin, and therefore for MP. However, detecting only the absolute amount of MP, the Raman response is attenuated to some degree by the combined absorption and scattering of anterior ocular media, predominantly the lens.
Accordingly, improved methods and apparatus that quickly, safely, and accurately measure a human's macular carotenoid levels are needed.